Methods And Compositions For Targeting Sequences Of Interest To The Chloroplast

ABSTRACT

Chimeric polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide of interest are provided, wherein the chloroplast transit peptide comprises an amino acid sequence having the chloroplast transit peptide sequence as set forth in SEQ ID NO:1 or a biologically active variant or fragment thereof or wherein the chloroplast transit peptide comprises the sequence set forth in SEQ ID NO: 58 or an active variant or fragment thereof. Chimeric polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the chimeric polynucleotides. Compositions further include HPPD polypeptides and polynucleotides encoding the same as set forth in SEQ ID NOS: 57 and 60 or active variants and fragments thereof. Such sequences comprise the chloroplast transit peptide as set forth in SEQ ID NO: 58 or an active variants or fragments thereof. Cells, plant cells, plants and seeds are further provided which comprise such sequences. Methods of use of the various sequences are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 61/401,456, filed Aug. 13, 2010; U.S. Provisional Ser. No. 61/393,507, filed Oct. 15, 2010; and, U.S. Provisional Ser. No. 61/501,042, filed Jun. 24, 2011; each of which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically, this invention pertains to targeting sequences of interest to a chloroplast by employing a novel chloroplast transit peptide.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 408355seqlist.txt, created on Aug. 12, 2011, and having a size of 108 KB and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety

BACKGROUND OF THE INVENTION

Plastids are a heterogeneous family of organelles found ubiquitously in plants and algal cells. Most prominent are the chloroplasts, which carry out such essential processes as photosynthesis and the biosynthesis of fatty acids as well as of amino acids. Chloroplasts are complex organelles composed of six distinct suborganellar compartments: three different membranes (the two envelope membranes and the internal thylakoid membranes) and three compartments (the innermembrane space of the envelope, the stroma and the thylakoid lumen.) More than 98% of all plastid proteins are translated on cytosolic ribosomes. Such proteins are posttranslationally targeted to and imported into the organelle. For a review, see, Jarvis et al. (2008) New Phytologist 179:257-285. Such translocation is mediated by multiprotein complexes in the outer and inner envelope membranes called TOC (Translocon at the Outer envelope membrane of Chloroplasts) and TIC (Translocon at the Inner envelope membrane of Chloroplasts). See, Soll et al. (2004) Nature Reviews. Molecular Cell Biology 5:198-208, Bedard et al. (2005) Journal of Experimental Botany 56:2287-2320, Kessler et al. (2006) Traffic 7:248-257, and Smith et al. (2006) Canadian Journal of Botany 84:531-542. Once the chloroplast precursor enters the stroma, the transit peptide if cleaved off, leaving the remaining part of the protein to take on its final confirmation or engage one of a number of different sorting pathways. See, Keegstra et al. (1999) Plant Cell 11:557-570, Jarvis et al. (2004) and Gutensohn et al. (2006) Journal of Plant Physiology 163:333-347.

Methods and compositions are needed to allow heterologous polypeptides to be targeted to the chloroplast.

BRIEF SUMMARY OF THE INVENTION

Chimeric polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide of interest are provided, wherein the chloroplast transit peptide comprises an amino acid sequence having the consensus monocot HPPD chloroplast transit peptide sequence as set forth in SEQ ID NO:1 or a biologically active variant or fragment thereof or wherein the chloroplast transit peptide comprises the sequence as set forth in SEQ ID NO: 58 or a biologically active variant or fragment thereof. Chimeric polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the chimeric polynucleotides. Methods of use of the various sequences are also provided.

Compositions further include novel HPPD polypeptides and polynucleotides encoding the same as set forth in SEQ ID NOS: 57 and 60 or active variants and fragments thereof. Such sequences comprise the chloroplast transit peptide as set forth in SEQ ID NO: 58 or an active variant or fragment thereof. Cells, plant cells, plants and seeds are further provided which comprise such sequences. Methods of use of the various sequences are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an amino acid alignment of HPPD from various monocot plants. HPPD from Hordeum vulgare is set forth in SEQ ID NO: 11. HPPD from Avena sativa is set forth in SEQ ID NO: 12. HPPD from Oryza sativa is set forth in SEQ ID NO:13. HPPD from Triticum aestivum is set forth in SEQ ID NO: 14. HPPD from Zea mays is set forth in SEQ ID NO: 10. HPPD from Sorghum bicolor is set forth in SEQ ID NO: 54. The underlining denotes amino acid residues sharing identity and the shading further displays the conserved amino acid residues.

FIG. 2 provides an amino acid alignment of HPPD polypeptides from various dicot plants compared to Zea mays SEQ ID NO: 10. HPPD from Daucus carota is set forth in SEQ ID NO: 15. HPPD from Solenostemon scutellarioides is set for in SEQ ID NO: 16. HPPD from Picea sitchenis is set for in SEQ ID NO: 17. HPPD from Abutilon theophrasti is set forth in SEQ ID NO: 18. HPPD from Arabidopsis thaliana is set forth in SEQ ID NO: 19. The HPPD from Brassica rapa is set forth in SEQ ID NO: 20. HPPD from Coptis japonica is set forth in SEQ ID NO: 21. HPPD from Vitis vinifera is set forth in SEQ ID NO: 22. HPPD from Glycine max is set forth in SEQ ID NO: 23. HPPD from Medicago truncatula is set forth in SEQ ID NO: 24.

FIG. 3 provides an alignment showing the diversity found in the N-terminal amino acids of HPPD polypeptides from moncot plants, dicot plants, microbes, a green alga and mammals.

FIG. 4A provides an alignment of the N-terminal amino acids of the HPPD polypeptide from various monocot plants. Amino acids 1-52 of the Zea mays HPPD are set forth in SEQ ID NO:3; amino acids 1-52 of the Sorghum bicolor HPPD are set forth in SEQ ID NO:4; amino acids 1-52 of the Oryza sativa HPPD are set forth in SEQ ID NO: 5; amino acids 1-48 of the Triticum aestivum HPPD are set forth in SEQ ID NO: 6; amino acids 1-46 of the Hordeum vulgare HPPD are set forth in SEQ ID NO:7; amino acids 1-47 of the Avena sativa HPPD are set forth in SEQ ID NO: 8; and the consensus sequence is set forth in SEQ ID NO: 2. FIG. 4B provides the % identity shared between the N-terminal regions of the HPPD polypeptides shown in FIG. 4A. The alignment was generated using AlignX which uses a modified Clustal W algorithm (program in Vector NTI (Invitrogen).)

FIG. 5A-C provides fluorescence microscopy of maize leaf tissue transfected with chloroplast-targeted or untargeted DsRed. FIG. 5A shows fluorescence observed in maize leaf transfected with ZmRCA1-Pro::RCA1CTP-Ds-Red2, 1000×. FIG. 5B shows fluorescence observed in maize leaf transfected with ZmRCA1-Pro::N-term-ZmHPPD-Ds-Red2, 1000×. FIG. 5C shows fluorescence observed in maize leaf transfected with untargeted Ds-Red2, 1000×. Photos on the left were of the same sample taken with white light.

FIG. 6 provides fluorescence microscopy of maize leaf tissue transformed by co-bombardment with plasmids coding for cycle 3 green fluorescence protein in combination with a plasmid coding for either Rubisco activase CTP fused to DsRed (A-D), the N-terminal 50 amino acids of maize HPPD fused to DsRed (E-G), or untargeted DsRed (H-J). The red channel (Figs. B, F and I) shows the pattern of DsRed fluorescence, the green channel (Figs. D, G, and J) cytosolic C3GFP fluorescence and the blue channel (Fig. C) chlorophyll autofluorescence. Overlays of the red and green channels are shown in figures A, E and H.

FIG. 7A-E provides an alignment of additional HPPD sequences.

FIG. 8 shows transient expression of Gm HPPD-AcGFP fusion proteins in soy leaf cells. Epifluorescence micrographs of soy leaf sections infiltrated with both untargeted (cytoplasmic) DsRed2 and Gm-HPPD N terminus fusions to AcGFP. A and C. With both vectors red fluorescence is seen in the cytoplasm while plastids remain dark. B. When AcGFP is fused to Gm-HPPD amino acids 42-86 (from SEQ ID NO: 57), green fluorescence is seen in the cytoplasm and plastids remain dark. D. When AcGFP is fused to Gm HPPD amino acids 1-86 (from SEQ ID NO: 57), green fluorescence is clearly seen in plastids of infected cells.

FIG. 9 shows that 50 amino acids of the maize HPPD N-terminus effectively targeted DsRed to plastids. N-terminal 0, 10, 20, 30, 40 or 50 amino acids of Zea Mays HPPD fused to Ds-Red. A-F: DsRed fluorescence micrographs A) 0aa, B) 10aa, C) 20aa, D) 30aa, E) 40aa F) 50aa.

FIG. 10 shows AcGFP fluorescence confocal micrograph of soybean leaf epidermal cell transiently expressing AcGFP linked to 50 amino acids of maize HPPD N-terminus in both the chloroplasts and cytoplasm.

FIG. 11 shows a leaf section of stably transformed soybean leaf showing sub-cellular localization of Z mays HPPD protein. CP: chloroplast; CY: cytosol; NUC: nucleus.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

I. Overview

In the production of transgenic plants it is often useful to direct foreign proteins to specific subcellular locations, e.g., the plastid, vacuole, mitochondria, or ER. When the gene is translated, the resulting protein has the transit peptide fused to the amino terminus of the protein of interest, and thus the protein is directed to the desired subcellular compartment. Of particular interest is the identification of transit peptides that will direct transport to a plastid. As used herein, a “plastid” refers to an organelle present in plant cells that stores and manufactures chemical compounds used by the cell, such as starch, fatty acids, terpenes, and that has been derived from a proplastid. Thus, plastids of plants typically have the same genetic content. Plastids include chloroplasts, which are responsible for photosynthesis, amyloplasts, chromoplasts, statoliths, leucoplasts, elaioplasts, and proteinoplasts. Plastids contain photosynthetic machinery and many additional biosynthetic enzymes including those leading to the production of fatty acids, amino acids, carotenoids, terpenoids, and starch. Thus, there is a need for the ability to target polypeptides of interest to plastids to modulate or alter the physiological processes that occur within these organelles. In addition, some polypeptides are toxic when expressed recombinantly in the cytoplasm. Because plastids are subcompartments, it is possible to target polypeptides of interest to the plastids to sequester them from the cytoplasm, and thus allow for higher expression levels. Furthermore, expression of recombinant polypeptides in plastids may facilitate isolation of the polypeptide for various applications. As discussed in further detail herein, novel CTP polypeptides from hydroxyphenylpyruvate dioxygenase polypeptides are provided which can be used in plastid targeting.

PSORT, a program that uses sequence data to predict organelle targeting, does not identify the N-terminal region of plant hydroxyphenylpyruvate dioxygenase (HPPD) proteins as a plastid targeting polypeptide. However, as demonstrated herein, HPPD polypeptides do contain a plastid targeting sequence which can be employed in a variety of methods and compositions to aid in targeting polypeptides of interest to the plastids. Thus, compositions and methods are provided for the targeting of polypeptides of interest to the chloroplast of a plant or plant cell.

The compositions provided herein include polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide (CTP) derived from an HPPD polypeptide operably linked to a nucleotide sequence encoding a polypeptide of interest. The CTP-encoding sequences disclosed herein, when assembled within a DNA construct such that the CTP-encoding sequence is operably linked to a nucleotide sequence encoding the polypeptide of interest, facilitate co-translational or post-translational transport of the peptide of interest to the chloroplast of a plant cell.

II. Chloroplast Transit Peptides

Chloroplasts are organelles found in plant cells and eukaryotic algae that conduct photosynthesis. The chloroplast is a complex cellular organelle composed of three membranes: the inner envelope membrane, the outer envelope membrane, and the thylakoid membrane. The membranes together enclose three aqueous compartments termed the intermediate space, the stroma, and the thylakoid lumen. While chloroplasts contain their own circular genome, many constituent chloroplast proteins are encoded by the nuclear genes and are cytoplasmically-synthesized as precursor forms which contain N-terminal extensions known as chloroplast transit peptides (CTPs). As used herein, the term “chloroplast transit peptide” or “CTP” refers to the N-terminal portion of a chloroplast precursor protein and is instrumental for specific recognition of the chloroplast surface and in mediating the post-translational translocation of pre-proteins across the chloroplast envelope and into the various subcompartments within the chloroplast (e.g. stroma, thylakoid and thylakoid membrane). Thus, as used herein, a polypeptide having “CTP activity” comprises a polypeptide which when operably linked to the N-terminal region of a protein of interest facilitates translocation of the polypeptide of interest to the chloroplast.

In one embodiment, a CTP is provided comprising the following HPPD CTP consensus sequence.

(SEQ ID NO: 1) MPPTP(T/A) (T/P/A) (T/P/A) (A/T) (G/T/A) (G/T/A) (G/A/*) (A/*) (G/V/*) (A/S/V) AA(A/S) (A/S/V) (T/A) (P/G/*)E(HN/Q) A(A/G/R) (F/P/R) (R/*)(L/*)(V/*)(G/S/*) (H/F/*)(R/H/P) (R/N)(F/M/V) VR(F/A/V) NPRSDRF (H/Q/P)(T/A/V)L(A/S)FHHVE or an active variant or fragment thereof, where the * indicates that that amino acid position is not represented (ie. a gap in the alignment).

In further embodiments, a synthetic consensus HPPD sequence comprising a CTP is provided having the following sequence:

(SEQ ID NO: 2) MPPTPTTAAATGAGAAAAVTPEHAAFRLVGHRRFVRFNPRSDRFH TLAFHHVE or an active variant or fragment thereof.

In still other embodiments, a CTP is provided that comprises the N-terminal region of any HPPD polypeptide, including for example, the N-terminal region of a monocot HPPD polypeptide or a dicot HPPD. In one embodiment, the CTP can comprise amino acids 1-53, 1-17, 1-19, 1-20, 1-23, 1-30, 1-40 and 1-60 or a variant or fragment thereof of any monocot HPPD polypeptide. For example, the CTP can comprise any one of SEQ ID NO:3 (amino acids 1-52 of the Zea mays HPPD); SEQ ID NO: 4 (amino acids 1-52 of the Sorghum bicolor HPPD); SEQ ID NO: 5 (amino acids 1-52 of the Oryza sativa HPPD); SEQ ID NO: 6 (amino acids 1-48 of the Triticum aestivum HPPD); SEQ ID NO:7 (amino acids 1-46 of the Hordeum vulgare HPPD); SEQ ID NO:8 (amino acids 1-47 of the Avena sativa HPPD); or an active variant or fragment of any one of SEQ ID NOS: 2, 3, 4, 5, 6, 7 or 8. The CTP-encoding sequence can further comprise any N-terminal region (about amino acids 1-53, 1-17, 1-19, 1-20, 1-30, 1-40 and 1-60 or 1-23) of any of the HPPD polypeptides as set forth in FIG. 2 or 7 or an active variant or fragment of such polypeptides. In addition, the CTP can comprise the sequence of SEQ ID NO:58 (amino acids 1-86 of the Soybean HPPD) or an active variant or fragment thereof.

It is recognized that the various CTPs disclosed herein can be modified to improve and/or alter the translocation of the polypeptide of interest into the chloroplast. For example, the CTP can contain additional regions that alter or improve the interactions with cytosolic factors that facilitate the passage of precursors from the ribosomes to the chloroplast surface. See, for example, Hiltbrunner et al. (2001) Journal of Cell Biology 154:309-316, Jackson-Constan et al. (2001) Biochimica et Biophysica Acta 1541:102-113, both of which are herein incorporated by reference. Other regions can be employed to increase the efficiency of chloroplast import. See, for example, May et al. (2000) Plant Cell 12:53-64, Qbadou et al. (2006) EMBO Journal 25:1837-1837 and Sohrt et al. (2000) Journal of Cell Biology 148:1213-1221, herein incorporated by reference. Such regions may be native (derived from a region of the HPPD polypeptide) or heterologous to the operably linked HPPD CTP.

The various CTP disclosed herein can further comprise additional sequences which modulate the final location of the polypeptide of interest in the chloroplast. For example, the various CTPs disclosed herein could further comprise a thylakoid lumen targeting domain. Proteins to be targeted to the thylakoid lumen bear an additional cleavable targeting signal, which like the transit peptide, is removed once translocation is complete. The luminal targeting peptides are extremely similar to the signal peptides that mediate inner membrane transport in bacteria. See, for example, Keegstra et al. (1999) Plant Cell 11:557-570, Jarvis (2004) Current Biology 14: R1064-R1077, Gutensohn et al. (2006) Journal of Plant Physiology 163:333-347, and Jarvis (2008) New Phytologist 179:257-285, all of which are incorporated by reference in their entirety, which discuss the various sorting pathways in a chloroplast. Such regions which modulate the location of the polypeptide of interest in a chloroplast may be native (derived from a region of the HPPD polypeptide) or heterologous to the operably linked HPPD CTP.

The term “chloroplast transit peptide cleavage site” refers to a site between two amino acids in a chloroplast-targeting sequence at which the chloroplast processing protease acts. CTPs target the desired protein to the chloroplast and can facilitate the protein's translocation into the organelle. This is accompanied by the cleavage of the transit peptide from the mature polypeptide or protein at the appropriate transit peptide cleavage site by a chloroplast processing protease. Accordingly, a CTP further comprises a suitable cleavage site for the correct processing of the pre-protein to the mature polypeptide contained within the chloroplast. In one non-limiting example, the CTP cleavage site is after amino acid 23, between Q and A, in SEQ ID NO:1 which would equate to the H/N/Q-A in SEQ ID NO: 1. As discussed above, the sequences beyond the cleaved fragments may be important for localization/transport efficiency and be employed with any of the CTPs disclosed herein.

The term “chimeric” sequence refers to a sequence having two or more heterologous sequences linked together. As used herein, a “heterologous” CTP comprises a transit peptide sequence which is foreign to the polypeptide of interest it is operably linked to. In one embodiment, the heterologous chloroplast transit peptide comprises any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 58 or an active variant or fragment thereof.

Assays to determine the efficiency by which the CTP sequences of the invention target a protein of interest to a chloroplast are known. See, for example, Mishkind et al. (1985) J of Cell Biol 100:226-234, which is herein incorporated by reference in its entirety. A reporter gene such as glucuronidase (GUS), chloramphenicol acetyl transferase (CAT), or green fluorescent protein (GFP) is operably linked to the CTP sequence. This fusion is placed behind the control of a suitable promoter, ligated into a transformation vector, and transformed into a plant or plant cell. Following an adequate period of time for expression and localization into the chloroplast, the chloroplast fraction is extracted and reporter activity assayed. The ability of the isolated sequences to target and deliver the reporter protein to the chloroplast can be compared to other known CTP sequences. See, de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780. Protein import can also be verified in vitro through the addition of proteases to the isolated chloroplast fraction. Proteins which were successfully imported into the chloroplast are resistant to the externally added proteases whereas proteins that remain in the cytosol are susceptible to digestion. Protein import can also be verified by the presence of functional protein in the chloroplast using standard molecular techniques for detection, by evaluating the phenotype resulting from expression of a chloroplast targeted protein, or by microscopy.

As used herein, an “isolated” or “purified” polynucleotide or polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A polypeptide that is substantially free of cellular material includes preparations of polypeptides having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the polypeptide of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

As used herein, polynucleotide or polypeptide is “recombinant” when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid.

For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A polypeptide expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example, a variant of a naturally occurring gene is recombinant.

a. Polynucleotide and Polypeptide Fragments and Variants of CTPs

Fragments and variants of the CTP-sequences are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain CTP activity and are thus capable of facilitating the translocation of a polypeptide of interest into the chloroplast of a plant. Alternatively, fragments of a polynucleotide that is useful as a hybridization probe generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 nucleotides or up to the full length CTP.

A fragment of polynucleotide that encodes a biologically active portion of a CTP-polypeptide will encode at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 60, 65, 70, 75, 80, 85 contiguous amino acids, or up to the total number of amino acids present in any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8 or 58 or any one of the N-terminal regions (about amino acids 1-17, 1-19, 1-20, 1-23, 1-30, 1-40 or about 1-53, 1-86) of the HPPD polypeptide as set forth in FIG. 2 or 7 or in any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8 or 58. Fragments of a CTP-encoding sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of an HPPD protein.

“Variant” protein is intended to mean a protein derived from the protein by deletion (i.e., truncation at the 5′ and/or 3′ end) and/or a deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, have CTP activity. Such variants may result from, for example, genetic polymorphism or from human manipulation.

For polynucleotides, a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5′ and/or 3′ end and/or a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the CTPs disclosed herein. Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis or gene synthesis but which still encode a CTP.

Biologically active variants of a CTP (and the polynucleotide encoding the same) will have at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 58 or to the N-terminal region (about 1-53, about 1-17, about 1-19, about 1-20, about 1-23, about 1-30, about 1-40, about 1-60, about 1-70, about 1-75, about 1-80, about 1-85) of the HPPD polypeptides as set forth in FIGS. 2 and 6 or in any one of SEQ ID NOS:1-8 or 58.

The CTP-sequences and the active variants and fragments thereof may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the CTPs can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different CTP-sequences can be manipulated to create a new CTP possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the CTP sequences disclosed herein and other known CTPs to obtain a new polynucleotide coding for a polypeptide with an improved property of interest, such as an improved efficiency of transport to the chloroplast. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

III. Sequence Comparisons

The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percent sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence or protein sequence.

(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polypeptide sequence, wherein the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polypeptides. Generally, the comparison window is at least 5, 10, 15, or 20 contiguous amino acids in length, or it can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polypeptide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. BLASTP protein searches can be performed using default parameters. See, blast.ncbi.nlm.nih.gov/Blast.cgi.

To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTP for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

In one embodiment, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity). When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percent sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percent sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percent sequence identity.

(e) Two sequences are “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acids substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) “A model of evolutionary change in proteins.” In “Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M.O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (http://www.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through http://www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

As used herein, similarity score and bit score is determined employing the BLAST alignment used the BLOSUM62 substitution matrix, a gap existence penalty of 11, and a gap extension penalty of 1. For the same pair of sequences, if there is a numerical difference between the scores obtained when using one or the other sequence as query sequences, a greater value of similarity score is selected.

IV. Polynucleotides/Polypeptides of Interest

Any polynucleotide of interest (i.e., the “polypeptide of interest”) may be used with the CTP-encoding sequences disclosed herein. Such polynucleotides/polypeptides of interest include, but are not limited to, herbicide-tolerance coding sequences, insecticidal coding sequences, nematicidal coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences modifying plant traits such as yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and/or utilization, and oil content and/or composition. More specific polynucleotides of interest for the present invention include, but are not limited to, genes that improve crop yield, polypeptides that improve desirability of crops, genes encoding proteins conferring resistance to abiotic stress, such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those conferring resistance to toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms. It is recognized that any polypeptides of interest can be operably linked to the CTP-encoding sequences of the invention and expressed in a plant, so long as the polypeptide encoded by the polynucleotide is functional in chloroplasts.

These nucleotide sequences of interest may encode proteins involved in providing disease or pest resistance. By “disease resistance” or “pest resistance” is intended that the plants avoid the harmful symptoms that are the outcome of the plant-pathogen interactions. Disease resistance and insect resistance genes such as lysozymes or cecropins for antibacterial protection, or proteins such as defensins, glucanases or chitinases for antifungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects are all examples of useful gene products.

“Pest” includes, but is not limited to, insects, fungi, bacteria, viruses, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera. Viruses include but are not limited to tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Nematodes include but are not limited to parasitic nematodes such as root knot, cyst, and lesion nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include but are not limited to Pratylenchus spp. Fungal pests include those that cause leaf, yellow, stripe and stem rusts.

An “herbicide resistance protein” or a protein resulting from expression of an “herbicide resistance-encoding nucleic acid molecule” includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein. Herbicide resistance traits may be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides, genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene), HPPD inhibitors (e.g, the HPPD gene) or other such genes known in the art. See, for example, U.S. Pat. Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, and 6,867,293, each of which is herein incorporated by reference.

Polynucleotides that improve crop yield include dwarfing genes, such as Rht1 and Rht2 (Peng et al. (1999) Nature 400:256-261), and those that increase plant growth, such as ammonium-inducible glutamate dehydrogenase. Polynucleotides that improve desirability of crops include, for example, those that allow plants to have a reduced saturated fat content, those that boost the nutritional value of plants, and those that increase grain protein. Polynucleotides that improve salt tolerance are those that increase or allow plant growth in an environment of higher salinity than the native environment of the plant into which the salt-tolerant gene(s) has been introduced.

Polynucleotides/polypeptides that influence amino acid biosynthesis include, for example, anthranilate synthase (AS; EC 4.1.3.27) which catalyzes the first reaction branching from the aromatic amino acid pathway to the biosynthesis of tryptophan in plants, fungi, and bacteria. In plants, the chemical processes for the biosynthesis of tryptophan are compartmentalized in the chloroplast. See, for example, US Pub. 20080050506, herein incorporated by reference. Additional sequences of interest include Chorismate Pyruvate Lyase (CPL) which refers to a gene encoding an enzyme which catalyzes the conversion of chorismate to pyruvate and pHBA. The most well characterized CPL gene has been isolated from E. coli and bears the GenBank accession number M96268. See, U.S. Pat. No. 7,361,811, herein incorporated by reference. Additional sequences of interest are discussed in more detail below.

a. Hydroxyphenylpyruvate Dioxygenase (HPPD) Polynucleotides and Polypeptides

In one embodiment, the CTP-encoding sequence is operably linked to a heterologous polynucleotide encoding a hydroxphenylpyruvate dioxygenase (HPPD) polypeptide. Various HPPD polypeptides and active variants and fragments thereof are known, as discussed below.

Hydroxyphenylpyruvate dioxygenase (HPPD) converts hydroxyphenylpyruvate, derived from the aromatic amino acid biosynthesis pathway, to homogentisate. In plants, homogentisate is a precursor of tocopherols and plastoquinones, an electron carrier essential in the biosynthesis of carotenoids. Consequently, when HPPD is inhibited by herbicide inhibitors, the plant can not protect itself from the radicals generated by light activation of chlorophyll. More specifically, inhibition of HPPD polypeptide leads to the depletion of protective pigments in the plant tissue resulting in bleaching of tissues which leaves the plants vulnerable to damage by light. HPPD inhibitors are an important class of herbicides. Transgenes that confer crop tolerance to HPPD inhibitors would be of significant value, especially for managing weed resistance to glyphosate.

As used herein, “Hydroxyphenylpyruvate dioxygenase” and “HPPD” “4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4-HPPD)” and “p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (p-OHPP)” are synonymous and refer to a non-heme iron-dependent oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. In organisms that degrade tyrosine, the reaction catalyzed by HPPD is the second step in the pathway. In plants, formation of homogentisate is necessary for the synthesis of plastoquinone, an essential redox cofactor, and tocopherol. The structures of various HPPD polypeptides are known. See, for example, FIG. 1 which provides the phylogenetic diversity of several monocot HPPD polypeptides, including sequences from Hordeum vulgare, Avena sativa, Oryza sativa, Triticum aestivum, and Zea mays. FIG. 2 provides the phylogenetic diversity of several dicot HPPD polypeptides including Daucus carota, Solenosteman sautellarioides, Picea sitchensis, Abutilon theophrasti, Arabidopsis thaliana, Brassica rapa, Coptis japonica, Vitis vinifera, Glycine max, and Medicago truncatula. HPPD polypeptides from microbes and mammals are also known and non-limiting examples of these sequences appear in FIG. 7.

Various variants of HPPD sequences are also known. See, for example, U.S. Provisional Application 61/401,456, filed Aug. 13, 2010, Compositions and Methods Comprising Sequences having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein incorporated by reference in it entirety. See, also, US 2003/0066102, WO97/49816, US 2010/0197503, U.S. Pat. No. 7,312,379, U.S. Pat. No. 6,768,044, U.S. Pat. No. 6,245,698, U.S. Pat. No. 6,268,549, and U.S. Pat. No. 6,118,050, the contents of each is herein incorporated by reference in its entirety. A review of the various structures of HPPD polypeptides from microbes, mammals and plants can be found, for example, in Moran et al. (2005) Archives of Biochemistry and Biophysics 433:117-128, herein incorporated by reference in its entirety.

As used herein, “hydroxyphenylpyruvate dioxygenase activity” or “HPPD activity” refers to the conversion of 4-hydroxyphenylpyruvate to homogentisate. As used herein, a polypeptide having “HPPD activity” comprises an HPPD polypeptide or an active variant or fragment thereof that retains sufficient HPPD activity such that (i) when expressed at sufficient levels in a cell that requires HPPD activity for viability, the HPPD polypeptide or active variant or fragment thereof exhibits sufficient HPPD activity to maintain viability of the cell in which it is expressed; or (ii) when expressed in a cell that requires HPPD activity for viability, the HPPD polypeptide or active variant or fragment thereof, when expressed in combination with one or more additional HPPD polypeptides results in the viability of the cell. Methods to determine such kinetic parameters (i.e., K_(m), k_(cat), k_(cat)/K_(m)) are known. See, for example, U.S. Provisional Application 61/401,456, filed Aug. 13, 2010 Compositions and Methods Comprising Sequences having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein incorporated by reference.

In order to provide plants with tolerance to commercially useful application rates of at least one desired HPPD inhibitor, it is advantageous to use polynucleotides which encode HPPD polypeptides having sufficient HPPD activity and having an insensitivity to inhibition by at least one or more HPPD inhibitor.

As used herein, an “HPPD inhibitor” comprises any compound or combinations of compounds which decrease the ability of HPPD to catalyze the conversion of 4-hydroxyphenylpyruvate to homogentisate. In specific embodiments, the HPPD inhibitor comprises a herbicidal inhibitor of HPPD. Non-limiting examples of HPPD inhibitors include, triketones (such as, mesotrione, sulcotrione, topramezone, and tembotrione); isoxazoles (such as, pyrasulfotole and isoxaflutole); pyrazoles (such as, benzofenap, pyrazoxyfen, and pyrazolynate); and benzobicyclon. Agriculturally acceptable salts of the various inhibitors include salts, the cations or anions of which are known and accepted in the art for the formation of salts for agricultural or horticultural use. See, for example, WO2005/053407 herein incorporated by reference.

The insensitivity of an HPPD inhibitor can be determined by assaying the insensitivity of a cell, a plant, a plant cell expressing the HPPD polypeptide or active fragment or variant thereof. In such instances, the cell, plant, or plant cell expressing an HPPD sequence displays an insensitivity to an HPPD inhibitor or to a combination of HPPD inhibitors when compared to a control cell, plant or plant cell not expressing the HPPD sequence. “Increased tolerance” to a herbicide is demonstrated when plants which display the increased tolerance to a herbicide are subjected to the HPPD inhibitor and a dose/response curve is shifted to the right when compared with that provided by an appropriate control plant. Such dose/response curves have “dose” plotted on the x-axis and “percentage injury”, “herbicidal effect” etc. plotted on the y-axis. Plants which are substantially “resistant” or “tolerant” to the herbicide exhibit few, if any, bleached, necrotic, lytic, chlorotic or other lesions and are not stunted, wilted or deformed when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to kill weeds in the field.

V. Novel Hydroxyphenylpyruvate Dioxygenase (HPPD) Sequences

Compositions are further provided comprising a novel HPPD polypeptide comprising the CTP set forth in SEQ ID NO: 58 and active variants and fragments thereof. In specific embodiments, such HPPD encoding sequences include the polynucleotide set forth in SEQ ID NO: 60 and the polypeptide set forth in SEQ ID NO: 57, and active variants and fragments thereof. Such polypeptides are capable of being transported into the chloroplast of a plant cell. In some embodiments, the polynucleotide set forth in SEQ ID NO: 60 or an active variant or fragment thereof is operably linked to a heterologous promoter.

In specific embodiments, active fragments and variants of the HPPD sequence as set forth in SEQ ID NO: 60 are provided. Such fragments comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,450 contiguous nucleotides, or up to the number of nucleotides present in SEQ ID NO: 60. Generally, variants of SEQ ID NO: 60 will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 60 as determined by sequence alignment programs and parameters described elsewhere herein. Active fragments and variants of SEQ ID NO: 60 will continue to encode a polypeptide having HPPD activity and which can be transported into the chloroplast of a plant cell.

The HPPD promoter as described in SEQ ID NO:1 of U.S. Provisional Application No. 61/501,042 leads to the production of at least two major transcripts from at least two transcription start sites (TSS1 and TSS2, see FIG. 5 of U.S. Provisional Application No. 61/501,042). The longer transcript initiates SEQ ID NO: 60 (encoding SEQ ID NO: 57). Parts of the genomic sequence transcribed to produce the longer transcript also act to promote transcriptional regulatory activity for the shorter transcript. Various polynucleotide sequences are known in the art which comprise multiple transcriptional start sites that encode products targeted to multiple cellular compartments. See for example, Small (1998) Plant Mol. Biol. 38:265-277 and Thatcher (2007) J of Biol. Chem. chloroplast, while the polypeptide encoded by the shorter transcript is localized to the cytosol.

Further provided are variant HPPD proteins as set forth in SEQ ID NO: 57. “Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, HPPD activity and wherein the protein is transported into the chloroplast of a plant cell. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a HPPD proteins disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 57 as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from SEQ ID NO: 57 by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Fragments of amino acid sequences include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a HPPD protein, or a partial-length protein and exhibiting HPPD activity but which include fewer amino acids than the full-length HPPD-related proteins disclosed herein. A biologically active portion of a HPPD protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200 contiguous amino acids in length, or up to the total number of amino acids present in a full-length HPPD protein of the current invention (i.e., of SEQ ID NO: 57). Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native HPPD protein, including but not limited to transport into the chloroplast of a plant cell. As used herein, a fragment comprises at least 5 contiguous amino acids of SEQ ID NO: 57. The invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, or 9 amino acids.

The polynucleotide encoding SEQ ID NO: 57 or active fragments and variants thereof can be provided in an expression cassette for expression in a plant or organism of interest. The expression cassette can include 5′ and 3′ regulatory sequences operably linked to the polynucleotide of the invention. An operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. In some embodiments, the polynucleotide set forth in SEQ ID NO: 60 can be operably linked to a heterologous promoter. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional polynucleotide to be cotransformed into the organism. Alternatively, the additional polypeptide(s) can be provided on multiple expression cassettes. Expression cassettes can be provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

Further provided are plants, plant cells, and seeds having a heterologous polynucleotide construct comprising an expression cassette having a promoter operably linked to a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 57 or an active variant or fragment thereof, wherein the promoter is heterologous to said polynucleotide.

VI. Plants

Plants, plant cells, plant parts and seeds, and grain having the polynucleotide comprising the CTP-encoding sequence operably linked to a heterologous polynucleotide encoding a polypeptide of interest are provided. In specific embodiments, the plants and/or plant parts have stably incorporated at least one of the chimeric polynucleotides disclosed herein or an active variant or fragment thereof. Thus, plants, plant cells, plant parts and seed are provided which comprise at least one polynucleotide comprising a CTP-encoding sequence operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein the chloroplast transit peptide comprises any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 58 or active variants and fragments thereof, or a CTP-encoding sequence of any one of the N-terminal regions (about amino acids 1-53, 1-20, 1-23, 1-17, 1-30, 1-40, 1-60, 1-70, 1-80, 1-85) of an HPPD polypeptide set forth in FIG. 2 or 7 or an active variant or fragment thereof. Further provided are plants, plant cells and seeds comprising the HPPD encoding sequences as set forth in SEQ ID NO: 57 and the polypeptide set forth in SEQ ID NO: 60, and active variants and fragments thereof.

Further provided are plants, plant cells, plant parts and seeds and grain having stably incorporated into their genome, the polynucleotide comprising a CTP-encoding sequence operably linked to a heterologous polynucleotide encoding a polypeptide of interest.

In specific embodiments, the chimeric polynucleotide or the HPPD encoding sequences in the plant or plant part is operably linked to a constitutive, tissue-preferred, or other promoter for expression in plants.

As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

The chimeric polynucleotides, the HPPD encoding sequences and active variant and fragments thereof disclosed herein may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifblia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include, but not limited to, tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include, but not limited to, azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pukherrima), and chrysanthemum.

Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

In some embodiments, the polynucleotides comprising the CTP-encoding sequence operably linked to the polynucleotide encoding the polypeptide of interest are engineered into a molecular stack. Thus, the various plants, plant cells and seeds disclosed herein can further comprise one or more traits of interest, and in more specific embodiments, the plant, plant part or plant cell is stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired combination of traits. As used herein, the term “stacked” includes having the multiple traits present in the same plant.

These stacked combinations can be created by any method including, but not limited to, breeding plants by any conventional methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.

A “subject plant or plant cell” is one in which genetic alteration, such as transformation, has been affected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

VII. Polynucleotide Constructs

The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

The chimeric polynucleotides or the HPPD encoding sequences disclosed herein can be provided in expression cassettes for expression in the plant of interest. The cassette can include 5′ and 3′ regulatory sequences operably linked to the chimeric polynucleotide or active variant or fragment thereof. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the chimeric polynucleotide or active variant or fragment thereof to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a CTP-encoding sequence or active variant or fragment thereof operably linked to a polynucleotide encoding a polypeptide of interest and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the CTP-encoding sequence and/or the polynucleotide encoding the polypeptide of interest may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the CTP-encoding sequence and/or the polynucleotide encoding the polypeptide of interest may be heterologous to the host cell or to each other. In specific embodiments, the CTP-encoding sequenced is operably linked to the 5′ end of the polynucleotide of interest, such that, in the resulting chimeric polypeptide, the CTP is operably linked to the N-terminal region of the polypeptide of interest.

As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked polynucleotide sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the CTP, the polynucleotide sequence of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used to express the various sequences of interest including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. Such promoters include, for example, constitutive, tissue-preferred, or other promoters for expression in plants.

Constitutive promoters include, for example, the core promoter of the Rsyn7/synthetic core II promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al, (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611. Additional promoters of interest are set forth in US Utility application Ser. No. ______ entitled “Chimeric Promoters And Methods of Use” filed concurrently herewith and herein incorporated by reference in its entirety.

Tissue-preferred promoters can be utilized to target enhanced HPPD expression within a particular plant tissue. Tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Synthetic promoters can be used to express the polynucleotide sequences of interest or biologically active variants and fragments thereof.

The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, sulfonylureas, dicamba, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention, including for example, DsRed as described in Example 3, 4, 6 or 9 and FIG. 5.

IIX. Method of Introducing

Various methods can be used to introduce a sequence of interest into a plant or plant part. “Introducing” is intended to mean presenting to the plant, plant cell or plant part the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant or plant part, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In specific embodiments, the polynucleotide comprising the CTP-encoding sequence operably linked to a heterologous polynucleotide encoding the polypeptide of interest or the sequence encoding the HPPD polypeptide can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the protein or active variants and fragments thereof directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference.

In other embodiments, the polynucleotide may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a DNA or RNA molecule. It is recognized that a protein sequence may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. Other methods to target polynucleotides are set forth in WO 2009/114321 (herein incorporated by reference), which describes “custom” meganucleases produced to modify plant genomes, in particular the genome of maize. See, also, Gao et al. (2010) Plant Journal 1:176-187.

The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

IX. Methods of Use

Methods of the present invention are directed to the proper expression, translocation, and processing of chloroplast-targeted sequences in plants and plant cells under the control of the CTP sequences disclosed herein. For the purposes of the present invention, a “processed” chloroplast targeted protein is one in which the CTP has been removed. At the time of translocation of a chloroplast targeted protein into the chloroplast of a plant cell, the CTP is removed from the targeted protein by cleavage at a particular “cleavage site” between the CTP and the mature protein. The cleavage site can be determined experimentally, or may be predicted based on sequence structure (e.g., by alignment of the unprocessed protein with chloroplast targeted proteins in which the cleavage site is known, by analyzing the sequence for the presence of characteristic CTP domains, and the like) or by using one or more algorithms for cleavage site prediction as discussed elsewhere herein (e.g., SignalP).

Thus, methods for targeting a polypeptide of interest to the chloroplast are provided. Such methods comprise introducing a chimeric polynucleotide comprising a CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest into a plant cell and expressing the chimeric polynucleotide in the plant cell.

Depending on the polypeptide of interest targeted to the chloroplast, the transgenic plants may have a change in phenotype, including, but not limited to, an altered pathogen or insect defense mechanism, an increased resistance to one or more herbicides, an increased ability to withstand stressful environmental conditions, a modified ability to produce starch, a modified level of starch production, a modified oil content and/or composition, a modified ability to utilize, partition and/or store nitrogen, and the like. These results can be achieved through the expression and targeting of a polypeptide of interest to chloroplasts in plants, wherein the polypeptide of interest functions in the chloroplast. The CTP sequences of the invention are useful for targeting native sequences as well as heterologous (non-native) sequences in plants.

X. Stacking Other Traits of Interest

In specific embodiments, the HPPD polynucleotides or active variants and fragments thereof disclosed herein or the various sequences encoding the chimeric polypeptides are engineered into a molecular stack. Thus, the various plants, plant cells and seeds disclosed herein can further comprise one or more traits of interest, and in more specific embodiments, the plant, plant part or plant cell is stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired combination of traits. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid, or both traits are incorporated into the genome of a plastid). In one non-limiting example, “stacked traits” comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. In one embodiment, the molecular stack comprises at least one additional polynucleotide that also confers tolerance to at least one HPPD inhibitor and/or at least one additional polynucleotide that confers tolerance to a second herbicide.

Thus, in one embodiment, the plants, plant cells or plant part having the HPPD polynucleotide or active variants or fragments thereof disclosed herein or a sequence encoding the chimeric polypeptides is stacked with at least one other HPPD sequence. Such HPPD sequence include the HPPD sequence and variants and fragment thereof disclosed herein, as well as other HPPD sequence, which include but are not limited to the HPPD sequences set forth in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115; international publication WO 99/23886, each of which is herein incorporated by reference, and those disclosed in U.S. Utility application Ser. No. ______ entitled “Compositions and Methods Comprising Sequences Having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity” filed concurrently herewith and incorporated by reference in its entirety.

In still other embodiments, plants, plant cells, explants and expression cassettes comprising the HPPD sequences, the various sequences encoding the chimeric polypeptides, or active variants and fragments thereof are stacked with a sequence that confers tolerance to HPPD inhibitors through a different mechanism than the HPPD polypeptide. For example, a P450 sequence could be employed which provides tolerance to HPPD-inhibitors by metabolism of the herbicide. Such sequences including, but are not limited to, the NSF1 gene. See, US 2007/0214515 and US 2008/0052797 both of which are herein incorporated by reference in their entirety.

Known genes that confer tolerance to herbicides such as e.g., auxin, HPPD, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides can be stacked either as a molecular stack or a breeding stack with plants expressing the traits disclosed herein. Polynucleotide molecules encoding proteins involved in herbicide tolerance include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 39,247; 6,566,587 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in U.S. Pat. Nos. 7,622,641; 7,462,481; 7,531,339; 7,527,955; 7,709,709; 7,714,188 and 7,666,643 also for providing glyphosate tolerance; dicamba monooxygenase disclosed in U.S. Pat. No. 7,022,896 and WO2007146706A2 for providing dicamba tolerance; a polynucleotide molecule encoding AAD12 disclosed in U.S. Pat. App. Pub. No. 2005731044 or WO2007053482A2 or encoding AAD1 disclosed in US20110124503A1 or U.S. Pat. No. 7,838,733 for providing tolerance to auxin herbicides (2,4-D); a polynucleotide molecule encoding hydroxyphenylpyruvate dioxygenase (HPPD) for providing tolerance to HPPD inhibitors (e.g., hydroxyphenylpyruvate dioxygenase) disclosed in e.g., U.S. Pat. No. 7,935,869; US20090055976A1; and US20110023180A1; each publication is herein incorporated by reference in its entirety.

In some embodiments, the plant or plant cells having the HPPD polynucleotides, the various sequences encoding the chimeric polypeptides or active variants or fragments thereof may be stacked with other herbicide-tolerance traits to create a transgenic plant of the invention with further improved properties. Other herbicide-tolerance polynucleotides that could be used in such embodiments include those conferring tolerance to glyphosate such as, for example, glyphosate N-acetyltransferase. See, for example, WO02/36782, US Publication 2004/0082770 and WO 2005/012515, U.S. Pat. No. 7,462,481, U.S. Pat. No. 7,405,074, each of which is herein incorporated by reference.

Additional glyphosate-tolerance traits include a sequence that encodes a glyphosate oxido-reductase enzyme as described more fully in U.S. Patent Nos. 5,776,760 and 5,463,175. Other traits that could be combined with the HPPD sequence disclosed herein include those derived from polynucleotides that confer on the plant the capacity to produce a higher level or glyphosate insensitive 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for example, as more fully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and international publications WO 97/04103; WO 00/66746; WO 01/66704; and WO 00/66747. Other traits that could be combined with the HPPD sequences disclosed herein include those conferring tolerance to sulfonylurea and/or imidazolinone, for example, as described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270.

In other embodiments, the plants or plant cell or plant part having the HPPD sequence or an active variant or fragment thereof is stacked with, for example, a sequence which confers tolerance to an ALS inhibitor. As used herein, an “ALS inhibitor-tolerant polypeptide” comprises any polypeptide which when expressed in a plant confers tolerance to at least one ALS inhibitor. A variety of ALS inhibitors are known and include, for example, sulfonylurea, imidazolinone, triazolopyrimidines, pryimidinyoxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinone herbicides. Additional ALS inhibitors are known and are disclosed elsewhere herein. It is known in the art that ALS mutations fall into different classes with regard to tolerance to sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidinyl(thio)benzoates, including mutations having the following characteristics: (1) broad tolerance to all four of these groups; (2) tolerance to imidazolinones and pyrimidinyl(thio)benzoates; (3) tolerance to sulfonylureas and triazolopyrimidines; and (4) tolerance to sulfonylureas and imidazolinones.

Various ALS inhibitor-tolerant polypeptides can be employed. In some embodiments, the ALS inhibitor-tolerant polynucleotides contain at least one nucleotide mutation resulting in one amino acid change in the ALS polypeptide. In specific embodiments, the change occurs in one of seven substantially conserved regions of acetolactate synthase. See, for example, Hattori et al. (1995) Molecular Genetics and Genomes 246:419-425; Lee et al. (1998) EMBO Journal 7:1241-1248; Mazur et al. (1989) Ann. Rev. Plant Phys. 40:441-470; and U.S. Pat. No. 5,605,011, each of which is incorporated by reference in their entirety. The ALS inhibitor-tolerant polypeptide can be encoded by, for example, the SuRA or SuRB locus of ALS. In specific embodiments, the ALS inhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALS mutant, the S4 mutant or the S4/HRA mutant or any combination thereof. Different mutations in ALS are known to confer tolerance to different herbicides and groups (and/or subgroups) of herbicides; see, e.g., Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Pat. Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which is herein incorporated by reference in their entirety. The soybean, maize, and Arabidopsis HRA sequences are disclosed, for example, in WO2007/024782, herein incorporated by reference.

In some embodiments, the ALS inhibitor-tolerant polypeptide confers tolerance to sulfonylurea and imidazolinone herbicides. The production of sulfonylurea-tolerant plants and imidazolinone-tolerant plants is described more fully in U.S. Patent Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270, which are incorporated herein by reference in their entireties for all purposes. In specific embodiments, the ALS inhibitor-tolerant polypeptide comprises a sulfonamide-tolerant acetolactate synthase (otherwise known as a sulfonamide-tolerant acetohydroxy acid synthase) or an imidazolinone-tolerant acetolactate synthase (otherwise known as an imidazolinone-tolerant acetohydroxy acid synthase).

In further embodiments, the plants or plant cell or plant part having the HPPD sequence or an active variant or fragment thereof is stacked with, for example, a sequence which confers tolerance to an ALS inhibitor and glyphosate tolerance. In one embodiment, the HPPD sequence or active variant or fragment thereof is stacked with HRA and a glyphosate N-acetyltransferase. See, WO2007/024782, 2008/0051288 and WO 2008/112019, each of which is herein incorporated by reference.

In still other embodiments, the plant or plant cell or plant part having the HPPD sequence or an active variant or fragment thereof may be stacked with, for example, aryloxyalkanoate dioxygenase polynucleotides (which confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in WO2005/107437) and dicamba-tolerance polynucleotides as described, for example, in Herman et al. (2005) J. Biol. Chem. 280: 24759-24767, auxin polypeptides and an acetyl coenzyme A carboxylase (ACCase) polypeptides.

Other examples of herbicide-tolerance traits that could be combined with the plant or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides or an active variants or fragments thereof include those conferred by polynucleotides encoding an exogenous phosphinothricin acetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903. Plants containing an exogenous phosphinothricin acetyltransferase can exhibit improved tolerance to glufosinate herbicides, which inhibit the enzyme glutamine synthase. Other examples of herbicide-tolerance traits that could be combined with the plants or plant cell or plant part having the HPPD sequence or an active variant or fragment thereof include those conferred by polynucleotides conferring altered protoporphyrinogen oxidase (protox) activity, as described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and international publication WO 01/12825. Plants containing such polynucleotides can exhibit improved tolerance to any of a variety of herbicides which target the protox enzyme (also referred to as “protox inhibitors”).

Other examples of herbicide-tolerance traits that could be combined with the plants or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides, or an active variant or fragment thereof include those conferring tolerance to at least one herbicide in a plant such as, for example, a maize plant or horseweed. Herbicide-tolerant weeds are known in the art, as are plants that vary in their tolerance to particular herbicides. See, e.g., Green and Williams (2004) “Correlation of Corn (Zea mays) Inbred Response to Nicosulfuron and Mesotrione,” poster presented at the WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12, 2004; Green (1998) Weed Technology 12: 474-477; Green and Ulrich (1993) Weed Science 41: 508-516. The trait(s) responsible for these tolerances can be combined by breeding or via other methods with the plants or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides or an active variants or fragments thereof to provide a plant of the invention as well as methods of use thereof.

In still further embodiments, the HPPD sequences, the various sequences encoding the chimeric polypeptides or active variants or fragments thereof can be stacked with at least one polynucleotide encoding a homogentisate solanesyltransferase (HST). See, for example, WO2010023911 herein incorporated by reference in its entirety. In such embodiments, classes of herbicidal compounds—which act wholly or in part by inhibiting HST can be applied over the plants having the HTS polypeptide.

The plant or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides, or an active variants or fragments thereof can also be combined with at least one other trait to produce plants that further comprise a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil content (e.g., U.S. Pat. No. 6,232,529); balanced amino acid content (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409; U.S. Pat. No. 5,850,016); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12:123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); the disclosures of which are herein incorporated by reference. Desired trait combinations also include LLNC (low linolenic acid content; see, e.g., Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH (high oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J. Agric. Food Chem. 53: 5326-5330).

The plant or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides or an active variants or fragments thereof can also be combined with other desirable traits such as, for example, fumonisim detoxification genes (U.S. Pat. No. 5,792,931), avirulence and disease resistance genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable for processing or process products such as modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine herbicide-tolerant polynucleotides with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.

In other embodiments, the plant or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides, or an active variants or fragments thereof may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109; Lee et al. (2003) Appl. Environ. Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) Acta Crystallogr. D. Biol. Crysiallogr. 57: 1101-1109 (Cry3Bbl); and Herman et al. (2004) J. Agric. Food Chem. 52: 2726-2734 (CrylF)), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825, pentin (described in U.S. Pat. No. 5,981,722), and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest.

In another embodiment, the plant or plant cell or plant part having the HPPD sequence, the various sequences encoding the chimeric polypeptides, or an active variant or fragment thereof can also be combined with the Rcg1 sequence or biologically active variant or fragment thereof. The Rcg1 sequence is an anthracnose stalk rot resistance gene in corn. See, for example, U.S. patent application Ser. No. 11/397,153, Ser. No. 11/397,275, and Ser. No. 11/397,247, each of which is herein incorporated by reference.

These stacked combinations can be created by any method including, but not limited to, breeding plants by any conventional methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.

Non-limiting embodiment include:

1. A chimeric polynucleotide comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein said chloroplast transit peptide comprises

a) an amino acid sequence comprising the amino acids of SEQ ID NO:1;

b) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58;

c) an amino acid sequence having at least 17 consecutive amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or 58; or,

d) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58 and having at least 17 consecutive amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or 58.

2. The chimeric polynucleotide of embodiment 1, wherein said chloroplast transit peptide comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58.

3. The chimeric polynucleotide of embodiment 1 or 2, wherein said polypeptide of interest comprises a 4-hydroxphenylpyruvate dioxygenase (HPPD) polypeptide having HPPD activity.

4. A nucleic acid construct comprising the chimeric polynucleotide of any one of embodiments 1-3.

5. The nucleic acid construct of embodiment 4, further comprising a promoter operably linked to said chimeric polynucleotide.

6. A cell comprising at least one chimeric polynucleotide of any of embodiments 1-3 or the nucleic acid construct of any one of embodiments 4 or 5.

7. The cell of embodiment 6, wherein said cell is a plant cell.

8. The cell of embodiment 7, wherein said polynucleotide or nucleic acid construct is stably incorporated into the genome of said plant cell.

9. The cell of any one of embodiments 7-8, wherein said plant cell is from a monocot.

10. The cell of embodiment 9, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.

11. The cell of any one of embodiments 7-8, wherein said plant cell is from a dicot.

12. The cell of embodiment 11, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

13. A plant comprising at least one plant cell of any one of embodiments 7-12.

14. A plant explant comprising at least one plant cell of any one of embodiments 7-12.

15. A transgenic seed produced by the plant of embodiment 13.

16. The plant, plant cell, or seed of any one of 11-15, wherein the plant, plant cell, or seed further comprises at least one polypeptide imparting tolerance to a herbicide.

17. The plant, plant cell, or seed of embodiment 16, wherein said at least one polypeptide imparting tolerance to a herbicide comprises:

-   -   (a) a sulfonylurea-tolerant acetolactate synthase;     -   (b) an imidazolinone-tolerant acetolactate synthase;     -   (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate         synthase;     -   (d) a glyphosate-tolerant glyphosate oxido-reductase;     -   (e) a glyphosate-N-acetyltransferase;     -   (f) a phosphinothricin acetyl transferase;     -   (g) a protoporphyrinogen oxidase.     -   (h) an auxin enzyme or receptor;     -   (i) a P450 polypeptide; or,     -   (j) an acetyl coenzyme A carboxylase (ACCase).

18. A chimeric polypeptide encoded by the polynucleotide of any one of embodiments 1-3.

19. A method of targeting a polypeptide of interest to a chloroplast comprising expressing a chimeric polynucleotide of any one of embodiments 1-3 or the nucleic acid construct of embodiment 4 or 5 in a plant cell.

20. A method of targeting a polypeptide of interest to a chloroplast comprising introducing the chimeric polynucleotide of any one of embodiments 1-3 or the nucleic acid construct of embodiment 4 or 5 in a plant cell and expressing said chimeric polynucleotide in the plant cell.

21. The method of embodiment 19 or 20, wherein said method further comprises regenerating a transgenic plant from said plant cell.

22. The method of any one of embodiments 19-21, wherein said plant cell is from a dicot.

23. The method of embodiment 22, wherein said dicot is selected from the group consisting of soybean, Brassica, sunflower, cotton, or alfalfa.

24. The method of any one of embodiments 19-21, wherein said plant cell is from a monocot.

25. The method of embodiment 24, wherein said dicot is selected from the group consisting of maize, wheat, rice, barley, sorghum, or rye.

26. The method of any one of embodiments 19-25, wherein the plant cell further comprises at least one polypeptide imparting tolerance to a herbicide.

27. The plant, plant cell, or seed of embodiment 26, wherein said at least one polypeptide imparting tolerance to a herbicide comprises:

-   -   (a) a sulfonylurea-tolerant acetolactate synthase;     -   (b) an imidazolinone-tolerant acetolactate synthase;     -   (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate         synthase;     -   (d) a glyphosate-tolerant glyphosate oxido-reductase;     -   (e) a glyphosate-N-acetyltransferase;     -   (f) a phosphinothricin acetyl transferase;     -   (g) a protoporphyrinogen oxidase.     -   (h) an auxin enzyme or receptor;     -   (i) a P450 polypeptide; or,     -   (j) an acetyl coenzyme A carboxylase (ACCase).

28. An expression cassette comprising a nucleic acid molecule operably linked to a heterologous promoter, wherein said heterologous promoter drives expression in a plant and wherein said nucleic acid molecule is selected from the group consisting of:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 60;

b) a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 60, wherein said nucleotide sequence encodes a polypeptide that has HPPD activity and is transported into the chloroplast;

c) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 57; and,

d) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 57, wherein said nucleotide sequence encodes a polypeptide that has HPPD activity and is transported into the chloroplast; and,

e) a complement of any of a)-d).

29. A plant cell comprising at least one expression cassette of embodiment 28.

30. The plant cell of embodiment 29, wherein said plant cell is a monocot.

31. The plant cell of embodiment 30, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.

32. The plant cell of embodiment 30, wherein said plant is from a dicot.

33. The plant cell of embodiment 32, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

34. A plant comprising at least one plant cell of any one of embodiments 29-33.

35. A transgenic seed produced by the plant of embodiment 34, wherein the seed comprises said expression cassette.

36. An isolated polypeptide selected from the group consisting of:

-   -   a) a polypeptide comprising the amino acid sequence of SEQ ID         NO:57; or,     -   b) a polypeptide comprising an amino acid sequence having at         least 90% sequence identity to the amino acid sequence of SEQ ID         NO:57, wherein said polypeptide has HPPD activity and is         transported into the chloroplast of a plant cell.

37. The plant, plant cell, or seed of any one of embodiments 29-35, wherein the plant, plant cell, or seed further comprises at least one polypeptide imparting tolerance to an additional herbicide.

38. The plant, plant cell, or seed of embodiment 37, wherein said at least one polypeptide imparting tolerance to an additional herbicide comprises:

-   -   (a) a sulfonylurea-tolerant acetolactate synthase;     -   (b) an imidazolinone-tolerant acetolactate synthase;     -   (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate         synthase;     -   (d) a glyphosate-tolerant glyphosate oxido-reductase;     -   (e) a glyphosate-N-acetyltransferase;     -   (f) a phosphinothricin acetyl transferase;     -   (g) a protoporphyrinogen oxidase.     -   (h) an auxin enzyme or receptor;     -   (i) a P450 polypeptide; or,     -   (j) an acetyl coenzyme A carboxylase (ACCase).

39. The plant, plant cell, or seed of embodiment 37, wherein said at least one polypeptide imparting tolerance to an additional herbicide comprises a high resistance allele of acetolactate synthase (HRA) and/or a glyphosate-N-acetyltransferase polypeptide.

EXPERIMENTAL Example 1 Maize HPPD has a Chloroplast Targeting Sequence Bioinformatic Analysis of Maize HPPD:

Maize HPPD proteins are not predicted to have a chloroplast targeting peptide N-terminal sequence by ProtComp 6.1 (http://linux1.softberry.com/berry.phtml), a widely used program for detecting organellar targeting sequences. ProtComp 6.1 indicates a cytosolic location of maize HPPD. The results returned by the search are as follows:

Significant similarity in Location: Cytoplasmic

Cytoplasmic score=14470

Chloroplastic score=1.4

Similarly, WoLF PSORT (Horton et al. (2007) NAR 35:W585-W587) and TargetP (Emanuelsson et al., (2000) J. Mol. Biol. 300:1005-1016) predict a cytosolic location of the HPPD protein. Protein Prowler (Hawkins and Boden (2006) J>Bioinf. Comp. Bio. 4:1-18) predicts either a mitochondrial (0.34) or chloroplast (0.39) location and Multiloc (Hoglund et. al. (2006) Bioinformatics 22:1158-1165) predicts an extracellular (0.74) localization with the first 50 amino acids of maize HPPD but a chloroplast localization (0.97) for the full maize HPPD sequence (SEQ ID NO 10). However, MultiLoc fails to predict a CTP function for the first N-terminal 50 amino acids of maize HPPD, suggesting that additional sequences may be important for full function.

Evaluation of the Glycine max HPPD protein (SEQ ID NO: 23) gave similarly variable results with the various prediction programs with predictions of peroxisomal, cytoplasmic, extracellular and chloroplast localization.

Example 2 Activity of Truncated Forms of Maize HPPD

Organellar targeting sequences are usually cleaved after the peptide enters the organelle. Previous investigators (Fritze I et al. (2004) Plant Physiology 134:1388-1400; Yang C et al. (2004) Biochemistry 43: 10414-10423) have shown that native mature maize HPPD begins at either ala17 or ala23. Variants of the wild-type maize HPPD protein coding region were created with various lengths of the amino terminus removed. The sequences were expressed in E. coli and tested for activity and stability. In each case a methionine start codon was added to the truncated sequence. Proteins were designated by the position of their N-terminal amino acid (all alanines) as in SEQ ID NO: 9. All N-terminal truncated proteins retained the HPPD activity. Differences in the measured k_(cat) may not be significantly different as only a single measurement was taken for this experiment. Assaying HPPD activity was carried out as described in Example 1 of Provisional Application 61/401,456, filed Aug. 13, 2010, Compositions and Methods Comprising Sequences having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein incorporated by reference in it entirety.

TABLE 1a Activity of N-terminal truncated- variants of maize wild-type HPPD. Truncation kcat, min−1 Maize wt 166 Ala12 230 Ala15 177 Ala17 180 Ala20 128 Ala23 184

Replicated data with two shuffled variants clearly showed that when the proteins were truncated such that their second amino acid (after the N-terminal methionine) is ala20, no significant differences in kinetic parameters were found.

TABLE 1b Kinetic parameters of variants truncated to ala20 Km, mM kcat, min−1 kcat/Km Full-length var A  6.61 ± 0.84 247 ± 47.1 37.2 ± 2.39 Truncated var. A  6.86 ± 0.37 206 ± 20.5 30.2 ± 4.64 Full-length var. B 11.80 ± 0.99 106 ± 12.4 9.00 ± 0.29 Truncated var. B 11.38 ± 0.96 93.3 ± 5.41  8.22 ± 0.36

To test stability, the variants were heated at various temperatures in the range of 20° C. to 54° C. for 30 minutes. The remaining activity was determined by the coupled assay described in Example 1 of Provisional Application 61/401,456, filed Aug. 13, 2010, Compositions and Methods Comprising Sequences having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein incorporated by reference in it entirety. All variants were stable at 20° C., but activity declined with incubation temperatures over 30° C. to nearly nil at 54° C. There were no differences in stability among wild-type and all truncated variants. Thus, maize HPPD does not require the N-terminal region of the protein for full enzymatic function in vitro.

Example 3 The N-Terminus of Maize HPPD Fused to DsRed is Targeted to Chloroplasts when Transiently Expressed in Maize Leaf

A vector was constructed in which the portion of the maize HPPD (SEQ ID NO: 9) gene coding for the N-terminal 50 amino acids was fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122) with a hygromycin selection cassette. Both genes are between left and right border sequences from Agrobacterium.

A positive control vector was identical to the vector having the N-terminal 50 amino acids of maize HPPD described above except that the HPPD CTP was removed and the DsRed2 insert was fused to the chloroplast targeting peptide of Zea maize rubisco activase, while a negative control was DsRed2 with no targeting sequence. The plasmids were transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration was used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila et. al. (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied.

Leaves of 3-week old maize seedlings were infiltrated with the Agrobacterium, and examined by fluorescence microscopy two days later (Nikon Eclipse 80i, DsRed filter set). With the vector where DsRed2 was fused to Rubisco activase CTP, the red fluorescence was seen in discrete packets in a pattern resembling peri-nuclear chloroplasts, as expected. A similar pattern was seen when DsRed2 was fused to the N-terminal 50 amino acids of maize HPPD. Without targeting, fluorescence was diffuse with some concentration in the nucleus. See FIG. 5.

In another experiment, maize leaf tissue was co-bombarded with DNA from both the DsRed-containing test plasmids and a plasmid encoding untargeted cycle 3 green fluorescence protein (C3GFP) using the PDS-1000 He biolistic particle delivery system (Bio-Rad, Hercules Calif.). Initial examination was conducted at approximately 24 h post-bombardment with a Lumar fluorescence stereomicroscope (Carl Zeiss Inc., Thornwood N.Y.) equipped with both a UV-exciting (Zeiss Set 01) and red-emitting (Zeiss Set 43 HE) filter set to image the C3GFP and the DsRed2, respectively. C3GFP fluorescence was captured using a 488 nm argon laser for excitation and a 500-550 nm band pass emission filter. DsRed fluorescence was imaged using a 561 nm diode laser for excitation and a 575-615 nm band pass emission filter. Chlorophyll fluorescence was captured by combining 561 nm excitation and a 650-710 nm band pass emission filter. The majority of maize leaf cells transformed were epidermal cells but because of the relatively low chlorophyll content of epidermal plastids it was difficult to verify plastid targeting of DsRed based on chlorophyll co-localization. Guard cell plastids, however, contained sufficient chlorophyll to be imaged via chlorophyll autofluorescence. Moderate to low-expressing guard cell pairs were chosen to illustrate plastid targeting (FIG. 6).

Transformation with vectors encoding RCA CTP-DsRed and the N-terminal 50 amino acids of maize HPPD fused to DsRed2 resulted in plastid targeting of the DsRed reporter. When fused to the known chloroplast targeting sequence of Rubisco activase, DsRed co-localized with chlorophyll autofluorescence (FIGS. 6B and 6C), whereas untargeted C3CFP showed no overlap with the Ds Red signal (FIG. 6A). Guard cell plastids could also be discerned by the exclusion of the C3GFP signal (FIGS. 6A, 6D, 6E, 6G and 6J) or the untargeted DsRed signal (FIGS. 6H and 6I). Plastid targeting of DsRed linked to the N-terminal 50 amino acids of maize HPPD was evident by a lack of overlap between the cytosolic C3GFP signal and the DsRed signal (Figs. F and G).

Example 4 0, 17, 20, 23, 30, 40 and 60 Amino Acid Fusions of Zea mays HPPD N-Terminal Region to Ds-Red and Visualization of Red Fluorescence in the Chloroplast

Vectors are constructed in which the portion of the maize HPPD gene (SEQ ID NO: 10) coding for the N-terminal 0, 17, 20, 23, 30, 40 or 60 amino acids are fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122) with a hygromycin selection cassette. Both genes are between left and right border sequences from Agrobacterium.

A positive control vector is identical except that the insert was DsRed2 fused to the chloroplast targeting peptide of Arabidopsis rubisco activase, while a negative control is DsRed2 with no targeting sequence. The plasmids are transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila, et. al., (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied.

Leaves of 3-week old maize seedlings are infiltrated with the Agrobacterium, and examined by fluorescence microscopy two days later (Nikon Eclipse 80i, DsRed filter set).

Example 5 Alignment of Monocot N-Terminal Regions to Show Similarity

FIG. 4 provides an N terminal alignment of monocot HPPD proteins with identities highlighted. The % identity table shows the relatedness of the fragments as shown. The proposed CTP activity is expected to be in the first 17-30 amino acids of each, although the sequences beyond the cleaved fragments may be important for localization. In view of the sequence of the monocot HPPD proteins, a consensus monocot HPPD chloroplast targeting peptide sequences was determined and provided in SEQ ID NO:1, where the * represents gaps in the alignment such that those position may be absent in a variant of the consensus sequence SEQ ID NO 2.

(SEQ ID NO: 1) MPPTP(T/A)(T/P/A)(T/P/A)(A/T)(G/T/A)(G/T/A)(G/A/*)(A/*)(G/V/*)(A/S/V)AA(A/S) (A/S/V)(T/A)(P/G/*)E(H/N/Q)A(A/G/R)(F/P/R)(R/*)(L/*)(V/*)(G/S/*)(H/F/*)(R/H/P) (R/N)(F/M/V)VR(F/A/V)NPRSDRF(H/Q/P)(T/A/V)L(A/S)FHHVE

A synthetic consensus monocot CTP from HPPD is further provided comprising the sequence set forth in SEQ ID NO: 2.

(SEQ ID NO: 2) MPPTPTTAAATGAGAAAAVTPEHAAFRLVGHRRFVRFNPRSDRFHTLAF HHVE.

Example 6 Fusion of Other Monocot N-Terminal Regions to Ds-Red and Visualization of Red Fluorescence in Maize Chloroplasts

Vectors are constructed in which N-terminal fragments (any amino acids from 1-20 or 1-60 or any region in between) of monocot HPPD proteins (SEQ ID NOS 10, 11, 12, 13, 14, 54) and the synthetic consensus peptide of SEQ ID NO: 2 is fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122) with a hygromycin selection cassette. Both genes are between left and right border sequences from Agrobacterium.

A positive control vector is identical except that the insert was DsRed2 fused to the chloroplast targeting peptide of Arabidopsis rubisco activase, while a negative control is DsRed2 with no targeting sequence. The plasmids are transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila et. al. (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied.

Leaves of 3-week old maize seedlings are infiltrated with the Agrobacterium, and examined by fluorescence microscopy two days later (Nikon Eclipse 80i, DsRed filter set).

Example 7 Recovery of Mature HPPD Protein from Maize Cells and Sequence of N-Terminus Showing the Cleavage Site after CTP Removal

Purified native HPPD was obtained from maize leaves by affinity chromatography on a column of immobilized anti-maize HPPD antibodies. Serum containing anti-maize HPPD antibodies was raised in rabbits antigenized with recombinant maize wild type 6×-his-HPPD produced in E coli and purified by nickel chelate affinity chromatography. The serum was passed through Protein A Ceramin Hyper DF to adsorb the IgG fraction. After washing, IgG was eluted with citrate buffer, pH 2.55, with a yield of 50 mg of IgG per gram of serum protein. Ten mg of IgG protein were subjected to the manufacturer's linkage protocol for Affi-Gel Hz (Bio-Rad), which resulted in the capture of 2 mg of IgG, 20% of which was anti-maize HPPD.

Maize leaf tissue was frozen in liquid nitrogen and pulverized in a mortar cooled with liquid nitrogen. The powder was mixed with 5 ml of 50 mM potassium phosphate, pH 7.3, 100 mM KCl, 10% ethylene glycol and 2 mM DTT. When thawed, the debris was removed by screening and the liquid centrifuged at 14,000×g, 15 min. The soluble protein fraction was desalted by passage through a gel-filtration column equilibrated with 50 mM potassium phosphate, 100 mM KCl, 10% ethylene glycol and the solution passed through the Affigel-anti-HPPD column. After extensive washing, pure native maize HPPD was eluted with 2 bed volumes of 0.5 M formic acid, then immediately neutralized. The preparation was subjected to Edman sequencing to determine the N-terminal sequence of mature maize HPPD protein.

Example 8 Function of Maize HPPD N-Terminal Sequence in Localizing Proteins to the Chloroplasts of Dicot Plant Cells

A vector is constructed in which the portion of the maize HPPD gene coding for the N-terminal 50 amino acids was fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the Arabidopsis Ubiquitin 10 promoter (Norris, et al. (1993) Plant Mol. Biol. 21, 895-906) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122) with a hygromycin selection cassette. Both genes are between left and right border sequences from Agrobacterium.

Leaf tissue of bush bean (common bean, Phaseolus vulgaris), are agro-infiltrated with Agrobacterium bacterial cell cultures of test and control strains. Infiltrated leaf samples are derived from plants of uniform developmental stage grown under the same conditions. Protoplasts are made from the infiltrated leaves 2-3 days after infection. Protoplasts can be isolated with proper osmoticum and enzyme digestion as by the method described by Rao and Prakash (1995) J. Biosci. 20:645-655. Protoplasts are examined using fluorescence microscopy using a Nikon Eclipse 80i, DsRed filter set to localize the N-terminal fusion proteins.

Example 9 Localization of Proteins Fused to the N-Terminal Fragments of Dicot HPPD Proteins

Vectors are constructed in which N-terminal fragments (20-60 amino acids) of dicot plant HPPD proteins (SEQ ID NOS 15-24) are fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) or the Arabidopsis Ubiquitin 10 promoter (Norris et al. (1993) Plant Mol. Biol. 21, 895-906) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122) with a hygromycin selection cassette. Both genes are between left and right border sequences from Agrobacterium. Such vectors can be used for either stable or transient gene expression in plant cells.

A positive control vector is identical except that the insert was DsRed2 fused to the chloroplast targeting peptide of Arabidopsis rubisco activase, while a negative control is DsRed2 with no targeting sequence. The plasmids are transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila, et. al., (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied.

Leaves of 3-week old maize seedlings are infiltrated with the Agrobacterium, and examined by fluorescence microscopy two days later (Nikon Eclipse 80i, DsRed filter set).

Leaf tissue of bush bean (common bean, Phaseolus vulgaris), are agro-infiltrated with Agrobacterium bacterial cell cultures of test and control strains. Infiltrated leaf samples are derived from plants of uniform developmental stage grown under the same conditions. Protoplasts are made from the infiltrated leaves 2-3 days after infection. Protoplasts can be isolated with proper osmoticum and enzyme digestion as by the method described by Rao and Prakash (1995) J. Biosci. 20:645-655. Protoplasts are examined using fluorescence microscopy using a Nikon Eclipse 80i, DsRed filter set to localize the N-terminal fusion proteins.

Example 10 Transformation and Regeneration of Transgenic Maize Plants

Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing an expression cassette comprising a CTP-encoding sequence disclosed herein operably linked to a polynucleotide encoding a polypeptide of interest operably linked to a promoter and the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.

A plasmid vector comprising the CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl₂ precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl₂; and, 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

The sample plates are bombarded at level #4 in a particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for the translocation of the polypeptide of interest to the chloroplast of the plant cell.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H₂O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H₂O), sterilized and cooled to 60° C.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H₂O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 11 Agrobacterium-Mediated Transformation of Maize Plants

For Agrobacterium-mediated transformation of maize with a CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest, the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring a CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.

Example 12 Soybean Embryo Stable Transformation

Soybean embryos are transformed with an expression cassette comprising a CTP-encoding sequence disclosed herein operably linked to a polynucleotide encoding a polypeptide of interest operably linked to a promoter and the selectable marker gene. Transformation is performed as follows.

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm, 26° C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures are transformed with the plasmids and DNA fragments by the method of particle gun bombardment (Klein et al. (1987) Nature, 327:70).

Soybean Embryogenic Suspension Culture Initiation

Soybean cultures are initiated twice each month with 5-7 days between each initiation.

Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 ml of autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mix well. Seeds are rinsed using 2 l-liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed are cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment

Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene are used for bombardment. Plasmid DNA for bombardment are routinely prepared and purified using the method described in the Promega™ Protocols and Applications Guide, Second Edition (page 106). Fragments of the plasmids carrying a CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest are obtained by gel isolation of double digested plasmids. In each case, 100 ug of plasmid DNA is digested in 0.5 ml of the specific enzyme mix that is appropriate for the plasmid of interest. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing a CTP-encoding sequence operably linked to a polynucleotide encoding a polypeptide of interest are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.

A 50 μl aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 5 μl of a 1 μg/0 DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 μl 2.5M CaCl₂ and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 μl 100% ethanol the pellet is suspended by sonication in 40 μl of 100% ethanol. Five μl of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μl aliquot contains approximately 0.375 mg gold per bombardment (i.e. per disk).

Tissue Preparation and Bombardment with DNA

Approximately 150-200 mg of 7 day old embryonic suspension cultures are placed in an empty, sterile 60×15 mm petri dish and the dish covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos

Transformed embryos were selected either using hygromycin (when the hygromycin phosphotransferase, HPT, gene was used as the selectable marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene was used as the selectable marker).

Hygromycin (HPT) Selection

Following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing a selection agent of 30 mg/L hygromycin. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.

Chlorsulfuron (ALS) Selection

Following bombardment, the tissue is divided between 2 flasks with fresh SB196 media and cultured as described above. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/ml Chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants

In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.

Embryo Maturation

Embryos are cultured for 4-6 weeks at 26° C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 uE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos can be removed from the clusters and screened for the presence of the polypeptide of interest in the chloroplast. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, could be screened at this stage.

Embryo Desiccation and Germination

Matured individual embryos are desiccated by placing them into an empty, small petri dish (35×10 mm) for approximately 4-7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they were left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then planted in Redi-Earth in 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10″ pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for proteins.

Media Recipes

SB 196 - FN Lite liquid proliferation medium (per liter) - MS FeEDTA - 100x Stock 1 10 ml MS Sulfate - 100x Stock 2 10 ml FN Lite Halides - 100x Stock 3 10 ml FN Lite P,B,Mo - 100x Stock 4 10 ml B5 vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L final concentration) 1.0 ml KNO₃ 2.83 gm (NH4)2 SO 4 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8

FN Lite Stock Solutions

Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x Stock Na₂ EDTA* 3.724 g 1.862 g FeSO₄—7H₂O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO₄—7H₂O 37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43 g CuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl₂—2H₂O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FN Lite P,B,Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 g Na₂MoO₄—2H₂O 0.025 g 0.0125 g *Add first, dissolve in dark bottle while stirring

SB1 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000× stock; 31.5 g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8 g TC agar.

SB 166 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose; 750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 g gelrite.

SB 103 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose; 750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.

SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL-Cat#21153-036); pH 5.7; and, 5 g TC agar.

2,4-D stock is obtained premade from Phytotech cat#D 295-concentration is 1 mg/ml.

B5 Vitamins Stock (per 100 ml) which is stored in aliquots at −20 C comprises: 10 g myo-inositol; 100 mg nicotinic acid; 100 mg pyridoxine HCl; and, 1 g thiamine. If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate. Chlorsulfuron Stock comprises 1 mg/ml in 0.01 N Ammonium Hydroxide.

Example 13 Glycine max HPPD has a Chloroplast Targeting Sequence

The G. max HPPD protein has been previously annotated as a 449 amino acid protein with N-terminal sequence MPIPMCNEIQ (SEQ ID NO:55) (See, U.S. Pat. No. 7,226,745 SEQ ID NO: 36) and as a 443 amino acid protein with N-terminal sequence MCNEIQAQAQ (SEQ ID NO: 56) (Genbank ABQ96868). Our analysis of G. max EST data revealed that an in-frame N-terminal extension of the previously annotated coding region exists, adding 41-amino acids to produce a 488 amino acid full-length HPPD protein (SEQ ID NO:57).

Bioinformatic and in-planta evaluation of the shorter G. max HPPD sequences did not reveal a chloroplast or other targeting sequence by prediction or by localization (see Example 1 and Example 9). The longer N-terminal region was able to direct a fluorescent marker protein to the chloroplast in dicot cells. See, Example 14 below. Bioinformatic analysis of the full-length G. max HPPD (SEQ ID NO: 57) revealed that a chloroplast targeting function is predicted. ProtComp 9.0 (http://linux1.softberry.com) returned the highest score for a membrane bound chloroplast localization based on the first 24 amino acids. WoLF P SORT (http://wolfpsort.org) also predicts a chloroplast location. TargetP (http://www.cbs.dtu.dk/services/TargetP) however only suggests a chloroplast localization, giving a higher score to ‘other’.

Example 14 Transient Expression of Gm HPPD-AcGFP Fusion Proteins

Numerous genes have been found to have two or more in-frame ATGs at the 5′ end. For review, see Small et al. (1998) Plant Molecular Biology 38: 265-277. Many of such genes are known to have multiple transcription starts to enable the production of two proteins from the same gene. Often, the “long” protein contains plastid targeting signal at the N-terminal while the “short” protein does not. Appropriate distribution of the “long” and “short” protein variants between two subcellular compartments is desired for the respective protein function to be carried out normally. The soy HPPD gene described here falls into this class of genes. No other HPPD gene is known to share the same description.

Transient expression experiments indicate that the long HPPD protein (SEQ ID NO: 57) is imported to chloroplasts, while the short protein (SEQ ID NO: 59) remains in the cytosol. Plant expression cassettes were constructed fusing portions of the N-terminus of Gm HPPD to an Aequorea coerulescens green fluorescent protein 1 (AcGFP 1). One fusion contained amino acid residues 1-86 of the long Gm HPPD protein. Another contained residues 1-44 of the short HPPD protein (this corresponds to residues 42-86 of the long protein). These cassettes were incorporated into binary vectors which also contained an untargeted DsRed2 expression cassette and introduced into A. tumefaciens strain AGL1 and then used to infect leaf discs of G. max as described in Example 3 of U.S. Utility application Ser. No. ______ entitled “Chimeric Promoter and Methods of Use”, filed concurrently herewith and herein incorporated by reference in its entirety. As shown in FIG. 8, green fluorescence is clearly visible in the chloroplasts of infected cells when AcGFP is fused to amino acid residues 1-86 of Gm HPPD. When the fusion is made with residues 42-86, corresponding to the 44 N-terminus residues of the short protein, green fluorescence is visible only in the cytoplasm.

Example 15 The N-Terminal 0, 10, 20, 30, 40 or 50 Amino Acids of Zea mays HPPD Fused to Ds-Red; Visualization of Red Fluorescence in the Z. Mays Chloroplast

Vectors were constructed in which the portion of the maize HPPD chloroplast targeting sequence (SEQ ID NO: 3) coding for the N-terminal 0, 10, 20, 30, 40 or 50 amino acids was fused to the gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into a binary expression vector under control of the maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) and terminated with the Solanum tuberosum proteinase inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell 1:115-122). The vector also contained an untargeted Zs Green cassette to provide cytoplasmic contrast and a kanamycin selection cassette. All three genes were between left and right border sequences of Agrobacterium T-DNA. A positive control vector was identical except that the insert was DsRed2 fused to the chloroplast targeting peptide of maize rubisco activase, while a negative control was DsRed2 with no targeting sequence. The plasmids were transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration was used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila, et. al., (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied. Leaves of 4-week old maize seedlings were infiltrated with the Agrobacterium, and examined by fluorescence microscopy four days later (Nikon Eclipse 80i, DsRed filter set).

Microscopy revealed that 50 amino acids of the maize HPPD N-terminus effectively targeted DsRed to plastids, but 40 amino acids or fewer failed to do so, with DsRed fluorescence visible only in the cytoplasm. See FIG. 9.

Example 16 Fusion of Other Monocot N-Terminal Regions to Ds-Red and Visualization of red fluorescence in maize and sorghum chloroplasts

A vector was constructed in which the monocot HPPD N-terminal consensus sequence (53 amino acids SEQ ID NO: 2) was fused to the gene coding for DsRed2 and assayed as described in Example 4. Microscopy revealed that the 53 amino acid consensus sequence effectively targeted DsRed to maize plastids, as did the maize rubisco activase positive control, but the untargeted negative control failed to do so, with DsRed fluorescence visible only in the cytoplasm.

This vector incorporating the 53 amino acid monocot HPPD N terminal consensus sequence targeting DsRed, and the 0, 10, 20, 30, 40, and 50 amino acid maize HPPD N-terminal (SEQ ID NO: 3) vectors described in Example 15 were also tested by Agro-infiltration in sorghum leaves. The results matched those obtained in maize; ie the 53 amino acid consensus sequence and the 50 amino acid maize sequence efficiently targeted the DsRed reporter protein to plastids, but shorter fragments failed to do so with red fluorescence visible only in the cytoplasm.

A vector was constructed in which the Oriza sativa HPPD N-terminal sequence (SEQ ID NO: 5) was fused to the gene coding for DsRed2 and assayed as described in example 4. Microscopy revealed that the 53 amino acid rice N-terminal sequence effectively targeted DsRed to maize plastids, as did the maize rubisco activase positive control, and the untargeted negative control failed to do so, with DsRed fluorescence visible only in the cytoplasm.

Example 17 Function of Maize HPPD N-Terminal Sequence in Localizing Proteins to the Chloroplasts of Dicot Plant Cells

The sequence encoding amino acids 1-50 of the maize HPPD protein (SEQ ID NO: 3) was fused to a gene encoding Aequorea coerulescens green fluorescent protein 1 (AcGFP1) and inserted into a binary expression vector under control of the Arabidopsis Ubiquitin 10 promoter (Norris et al. (1993) Plant Mol. Biol. 21, 895-906) and terminated with the Glycine max Kunitz trypsin inhibitor 3 terminator region (NCBI accession S45092). Both genes were between left and right border sequences from Agrobacterium. Such vectors can be used for either stable or transient gene expression in plant cells. A positive control vector was identical except that the AcGFP1 coding region was fused to the 6H1 synthetic chloroplast targeting peptide (U.S. Pat. No. 7,345,143 SEQ ID NO:1), while a negative control was AcGFP1 with no targeting sequence. The plasmids were transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila, et. al., (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived trans-gene expression may be measured or studied. Infiltrated leaf samples were derived from plants of uniform developmental stage grown under the same conditions. Leaves of 4-week old Nicotiana benthamiana, 8-day old Phaseolus vulgaris, and 10-day old Glycine max seedlings were infiltrated with the Agrobacterium, and examined by fluorescence microscopy 4 and 5 days later (Nikon Eclipse 80i, DsRed filter set). The first 50 amino acids of maize HPPD were sufficient to drive chloroplast import of AcGFP in epidermal cells of P. vulgaris, although some green fluorescence remained in the cytoplasm. In N. benthamiana the AcGFP remained in the cytoplasm with none apparent in the chloroplasts. Results in G. max showed AcGFP in both plastids and cytoplasm. When similar constructs were introduced to soybean leaf epidermal cells and examined by confocal microscopy, green fluorescence was apparent in the cytosol and chloroplasts of transformed cells, with variable intensity in chloroplasts (see FIG. 10). This shows that the maize HPPD CTP is recognized in dicot plant cells.

Example 18 Localization of Z. Mays HPPD Protein in Stably Transformed Soybean Cells

Polyclonal antibodies were raised in rabbits against recombinant maize HPPD protein purified by nickel chelate affinity chromatography. Anti-HPPD antibodies were purified from serum by affinity chromatography on immobilized maize HPPD, and further purified by passage through a column of immobilized Rubisco, to remove a small fraction of antibodies that reacted with both HPPD and Rubisco. Leaf punches taken form stably transformed soybean plants expressing a gene encoding the maize HPPD protein (SEQ ID NO: 10) driven by an SCP1 synthetic promoter (U.S. Pat. No. 6,072,050) were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde in 100 mM Na phosphate buffer, pH 7.0, for 3 hours at room temperature, dehydrated by passage through progressively higher concentrations of ethanol, embedded in LR White resin and cured at 55° C. for 48 hours. Sections (0.9 microns) were transferred onto Excell Adhesion glass microscope slides (Electron Microscopy Sciences). Immunolocalization was performed with the primary antibody being the double-purified anti-maize HPPD (1:200) and the secondary antibody goat anti-rabbit F(ab′) conjugated with gold particles (Aurion ultrasmall gold). Gold labeling was followed by silver enhancement (Aurion R-GENT SE-EM). Sections were counterstained with 4% uranyl acetate (aqueous) followed by Reynold's lead citrate. Material was analyzed with a YAG detector for backscatter signal in a Hitachi 54800 scanning electron microscope. The clarity of the resulting images was enhanced by performing contrast inversion, using Adobe PhotoShop CS5.

Gold labeling was observed mainly in cytosol and nuclei, but also in chloroplasts. See, FIG. 11. A small number of particles observed in other locations including voids are considered to be artifacts. This is consistent with transient expression experiments showing that the first 50 amino acids of the Z. mays HPPD N terminus did target a fluorescent reporter protein to the chloroplasts of G. max cells (see Example 17).

Example 19 G. max N-Terminus Targets Proteins to the Chloroplast in Maize Cells

Vectors were constructed in which a portion of the gene coding for the G. max HPPD N-terminus was fused to a gene encoding Aequorea coerulescens green fluorescent protein 1 (AcGFP1) and inserted into a binary expression vector under control of the Arabidopsis Ubiquitin 10 promoter (Norris et al. (1993) Plant Mol. Biol. 21, 895-906) and terminated with the Glycine max Kunitz trypsin inhibitor 3 terminator region (NCBI accession S45092). Both genes were between left and right border sequences from Agrobacterium. One fusion contained amino acid residues 1-86 of the long Gm HPPD protein (SEQ ID NO:57). Another contained residues 1-44 of the short HPPD protein (this corresponds to residues 42-86 of the long protein and SEQ ID NO:59).

A positive control vector was identical except that the AcGFP1 coding region was fused to the 6H1 synthetic chloroplast targeting peptide (U.S. Pat. No. 7,345,143), while a negative control was AcGFP1 with no targeting sequence. The plasmids were transformed into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to introduce the constructs into plant cells. Agro-infiltration is a well described method (Kapila et. al. (1997) Plant Science, 122:101-108) of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant trans-gene expression may be measured or studied.

Leaves of 4-week old maize seedlings were infiltrated with the Agrobacterium, and examined by fluorescence microscopy three days later (Nikon Eclipse 80i, Narrow band-pass GFP filter set). Transient expression indicated that the long G. max HPPD protein N-terminus (SEQ ID NO: 58) did target the marker protein to maize cell chloroplasts, while the short protein N-terminus (amino acids 1-44 of SEQ ID NO: 59) delivered the protein to the cytosol. The dicot chloroplast targeting region of G. max HPPD is able to function in monocot cells.

TABLE 2 Summary of SEQ ID NOS SEQ ID NO Description 1 Consensus Monocot CTP from HPPD 2 Synthetic consensus CTP from monocot HPPD 3 CTP of Zea mays HPPD 4 CTP of sorghum bicolor HPPD 5 CTP of Oryza sativa HPPD 6 CTP of Triticum aestivum HPPD 7 CTP of Hordeum vulgare HPPD 8 CTP of Avena Sativa HPPD 9 Full length Zea mays HPPD 10 Maize WT HPPD (from WO1997049816 SEQ ID NO: 11) 11 HPPD from Hordeum vulgare 12 HPPD from Avena sativa 13 HPPD from Oryza sativa 14 HPPD from Triticum aestivum 15 HPPD from Daucus carota 16 HPPD from Solenosteman sautellarioides 17 HPPD from Picea sitchensis 18 HPPD from Abutilon theophrasti 19 HPPD from Arabidopsis thaliana 20 HPPD from Brassica rapa 21 HPPD from Coptis japonica 22 HPPD from Vitis vinifera 23 HPPD from Glycine max 24 HPPD from Medicago truncatula 26-53 N-terminal regions of various HPPD polypeptides 54 HPPD from Sorghum bicolor 55 N-term amino acids of soy HPPD disclosed in U.S. Pat. No. 7,226,745 as SEQ ID NO: 36 56 N-term amino acids of soy HPPD disclosed in Genbank ABQ96868 57 Full length soybean HPPD 58 N-terminal region of SEQ ID NO: 57 comprising native soybean CTP 59 Soybean HPPD protein predicted from shorter transcript 60 Nucleotide sequence of the Full length soybean HPPD

The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 

That which is claimed:
 1. A chimeric polynucleotide comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein said chloroplast transit peptide comprises a) an amino acid sequence comprising the amino acids of SEQ ID NO:1; b) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58; c) an amino acid sequence having at least 17 consecutive amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or 58; or, d) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58 and having at least 17 consecutive amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or
 58. 2. The chimeric polynucleotide of claim 1, wherein said chloroplast transit peptide comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or
 58. 3. The chimeric polynucleotide of claim 1, wherein said polypeptide of interest comprises a 4-hydroxphenylpyruvate dioxygenase (HPPD) polypeptide having HPPD activity.
 4. A nucleic acid construct comprising the chimeric polynucleotide of claim
 1. 5. The nucleic acid construct of claim 4, further comprising a promoter operably linked to said chimeric polynucleotide.
 6. A cell comprising at least one chimeric polynucleotide of claim
 1. 7. The cell of claim 6, wherein said cell is a plant cell.
 8. The cell of claim 7, wherein said polynucleotide or nucleic acid construct is stably incorporated into the genome of said plant cell.
 9. The cell of claim 7, wherein said plant cell is from a monocot.
 10. The cell of claim 9, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
 11. The cell of claim 7, wherein said plant cell is from a dicot.
 12. The cell of claim 11, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
 13. A plant comprising at least one plant cell of claim
 7. 14. A plant explant comprising at least one plant cell of claim
 7. 15. A transgenic seed produced by the plant of claim
 13. 16. The plant cell of claim 7, wherein the plant cell further comprises at least one polypeptide imparting tolerance to a herbicide.
 17. The plant cell of claim 16, wherein said at least one polypeptide imparting tolerance to a herbicide comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase; (d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A carboxylase (ACCase).
 18. A chimeric polypeptide encoded by the polynucleotide of claim
 1. 19. A method of targeting a polypeptide of interest to a chloroplast comprising expressing a chimeric polynucleotide of claim 1 or the nucleic acid construct of claim 4 in a plant cell.
 20. A method of targeting a polypeptide of interest to a chloroplast comprising introducing the chimeric polynucleotide of claim 1 or the nucleic acid construct of claim 4 in a plant cell and expressing said chimeric polynucleotide in the plant cell.
 21. The method of claim 19, wherein said method further comprises regenerating a transgenic plant from said plant cell.
 22. The method of claim 19, wherein said plant cell is from a dicot.
 23. The method of claim 22, wherein said dicot is selected from the group consisting of soybean, Brassica, sunflower, cotton, or alfalfa.
 24. The method of claim 19, wherein said plant cell is from a monocot.
 25. The method of claim 24, wherein said dicot is selected from the group consisting of maize, wheat, rice, barley, sorghum, or rye.
 26. The method of claim 19, wherein the plant cell further comprises at least one polypeptide imparting tolerance to a herbicide.
 27. The method of claim 26, wherein said at least one polypeptide imparting tolerance to a herbicide comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase; (d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A carboxylase (ACCase).
 28. An expression cassette comprising a nucleic acid molecule operably linked to a heterologous promoter, wherein said heterologous promoter drives expression in a plant and wherein said nucleic acid molecule is selected from the group consisting of: a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 60; b) a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 60, wherein said nucleotide sequence encodes a polypeptide that has HPPD activity and is transported into the chloroplast; c) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 57; and, d) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 57, wherein said nucleotide sequence encodes a polypeptide that has HPPD activity and is transported into the chloroplast; and, e) a complement of any of a)-d).
 29. A plant cell comprising at least one expression cassette of claim
 28. 30. The plant cell of claim 29, wherein said plant cell is a monocot.
 31. The plant cell of claim 30, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
 32. The plant cell of claim 30, wherein said plant is from a dicot.
 33. The plant cell of claim 32, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
 34. A plant comprising at least one plant cell of claim
 29. 35. A transgenic seed produced by the plant of claim 34, wherein the seed comprises said expression cassette.
 36. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:57; or, b) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:57, wherein said polypeptide has HPPD activity and is transported into the chloroplast of a plant cell.
 37. The plant cell of claim 29, wherein the plant, plant cell, or seed further comprises at least one polypeptide imparting tolerance to an additional herbicide.
 38. The plant cell of claim 37, wherein said at least one polypeptide imparting tolerance to an additional herbicide comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase; (d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A carboxylase (ACCase).
 39. The plant cell of claim 37, wherein said at least one polypeptide imparting tolerance to an additional herbicide comprises a high resistance allele of acetolactate synthase (HRA) and/or a glyphosate-N-acetyltransferase polypeptide. 